The differential protein expression profiles and immunogenicity of tachyzoites and bradyzoites of in vitro cultured <Emphasis Type="Italic">Neospora caninum</Emphasis>

We report a study on the variations in the protein expression profiles of tachyzoites and bradyzoites of Neospora caninum. The in vitro stage conversion of N. caninum-infected Vero cells was induced by continuous treatment of infected cultures with 70 muM sodium nitroprusside (SNP) for up to 9 days. The stage conversion indicated by the expression of the bradyzoite-specific antigen BAG1 was analyzed by immunofluoresence assay. Morphological changes between tachyzoites and bradyzoites and localization of nuclei were demonstrated by transmission electron microscopy. Notably, we showed the differential protein expression profiles of tachyzoites and bradyzoites of N. caninum upon treatment with SNP. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated different protein patterns between tachyzoites and bradyzoites. Furthermore, Western blotting using rabbit polyclonal antibodies directed against tachyzoites revealed several reactive bands, one of which represented a tachyzoite-specific antigen of approximately 40 kDa remarkably expressed in the tachyzoite stage, but was absent from bradyzoites. Moreover, rabbit polyclonal serum raised against bradyzoites recognized a significant increased expression of an antigen with a MW of approximately 25 kDa in bradyzoites by Western blotting, suggesting that this protein is specifically expressed at the bradyzoite stage. Taken together, our data showed that differential protein expression profiling is a useful tool for discriminating between the two stages during tachyzoite-bradyzoite interconversion in N. caninum infections.     

Triclosan inhibits the growth of Neospora caninum in vitro and in vivo

Neospora caninum is an apicomplexan parasite considered one of the main causes of abortion in cattle worldwide; thus, there is an urgent need to develop novel therapeutic agents to control the neosporosis. Enoyl acyl carrier protein reductase (ENR) is a key enzyme of the type II fatty acid synthesis pathway (FAS II), which is essential for apicomplexan parasite survival. The antimicrobial agent triclosan has been shown to be a very potent inhibitor of ENR. In this study, we identified an E. coli ENR-like protein in N. caninum. Multiple sequence alignment showed all the requisite features of ENR existed in this protein, so we named this protein NcENR. Swiss-Model analysis showed NcENR interacts with triclosan. We observed that ENR is localized in the apicoplast, a plastid-like organelle. Similar to the potent inhibition of triclosan on other apicomplexa parasites, this compound markedly inhibits the growth of N. caninum at low concentrations. Further research showed that triclosan attenuated the invasion ability and proliferation ability of N. caninum at low concentrations. The results from in vivo studies in the mouse showed that triclosan attenuated the virulence of N. caninum in mice mildly and reduced the parasite burden in the brain significantly. Taken together, triclosan inhibits the growth of N. caninum both in vitro and in vivo at low concentrations.

     

Reevaluation of the Toxoplasma gondii and Neospora caninum genomes reveals misassembly,karyotype differences,and chromosomal rearrangements

Neospora caninum primarily infects cattle, causing abortions, with an estimated impact of a billion dollars on the worldwide economy annually. However, the study of its biology has been unheeded by the established paradigm that it is virtually identical to its close relative, the widely studied human pathogen Toxoplasma gondii. By revisiting the genome sequence, assembly, and annotation using third-generation sequencing technologies, here we show that the N. caninum genome was originally incorrectly assembled under the presumption of synteny with T. gondii. We show that major chromosomal rearrangements have occurred between these species. Importantly, we show that chromosomes originally named Chr VIIb and VIII are indeed fused, reducing the karyotype of both N. caninum and T. gondii to 13 chromosomes. We reannotate the N. caninum genome, revealing more than 500 new genes. We sequence and annotate the nonphotosynthetic plastid and mitochondrial genomes and show that although apicoplast genomes are virtually identical, high levels of gene fragmentation and reshuffling exist between species and strains. Our results correct assembly artifacts that are currently widely distributed in the genome database of N. caninum and T. gondii and, more importantly, highlight the mitochondria as a previously oversighted source of variability and pave the way for a change in the paradigm of synteny, encouraging rethinking the genome as basis of the comparative unique biology of these pathogens.

The Apicomplexa comprise a large phylum of parasitic alveolates of medical and veterinary importance, causing deadly diseases such as malaria, cryptosporidiosis, neosporosis, and toxoplasmosis, among others. With the exception of a few commonalities such as their obligatory intracellular lifestyle and the presence of specialized secretory organelles and of secondary endosymbionts, the apicomplexans differ greatly in morphology, host range specificity, pathogenicity, reproductive strategy, and transmission. Understanding the molecular basis of these differences has been the focus of much research. Comparative genomic analyses revealed that, albeit all small, apicomplexans genomes vary greatly in size, ranging from 9 to 130 Mb (Debarry and Kissinger 2011; Blazejewski et al. 2015). Having diverged from a common ancestor 350–824 myr ago (Escalante and Ayala 1995), shy of 900 genes are conserved among them, whereby major genomic rearrangements can be observed (Debarry and Kissinger 2011).High synteny, defined as conserved content and order of a given genomic locus, is rarely observed (Debarry and Kissinger 2011). A seemingly stark exception to this is the genomes of Toxoplasma gondii and Neospora caninum. Morphologically, these parasites are virtually indistinguishable, so much so, that N. caninum was only recognized as a separate species in 1988 (Dubey 2003; Dubey et al. 2002). Moreover, both species show similar tropism within their hosts, where they can infect virtually any nucleated cell. They both show a fast-replicating form (tachyzoite), causing acute disease, that transitions into a slow-dividing form (bradyzoite), which persists in immune-privileged sites, such as the brain, establishing chronic infection. In line with this, initial comparative analysis concluded that these species have largely conserved genomic content and are largely syntenic (Reid et al. 2012). Despite their commonalities, however, the biology of these pathogens also differs significantly. T. gondii infects a wide range of intermediate hosts, including humans, causing deadly disease in immunocompromised individuals or by congenital transmission. In contrast, N. caninum infects primarily cattle, causing abortions with an estimated impact of a billion dollars on the worldwide economy annually (Reichel et al. 2013). Feline species act as definitive hosts of T. gondii, whereas sexual replication of N. caninum occurs only in canids (McAllister et al. 1998; Gondim et al. 2004; King et al. 2010). These biological differences have been largely ascribed to absence, point mutations, and pseudogenization of T. gondii virulence factors in N. caninum and the comparative amplification of surface protein-coding gene families in N. caninum (Khan et al. 2009; Reid et al. 2012).Advancements in genome sequencing technologies have accompanied the fast-paced genomics era. Particularly, third-generation sequencing technologies, such as Pacific Biosciences (PacBio) and Oxford Nanopore Technologies sequencing, outperform prior technologies by providing very long reads that can span regions containing repetitive sequences. This has led to improvements in the assembly of previously unattainable genomes, such as those presenting high proportions of repetitive sequences, allowing whole new genomes to be assembled with high accuracy. Here, we set out to sequence and de novo assemble two N. caninum strain genomes and the T. gondii genome, using PacBio and Oxford Nanopore.     

Mycophenolic acid induces differentiation of Toxoplasma gondii RH strain tachyzoites into bradyzoites and formation of cyst-like structure in vitro

Parasitology Research - The biochemical and structural changes that occur during the conversion of Toxoplasma gondii tachyzoites to bradyzoites and the formation of tissue cyst are not well...     

Subcellular localization and functional characterization of Nc-p43, a major Neospora caninum tachyzoite surface protein.

Neospora caninum is a recently identified coccidian parasite which shares many features with, but is clearly distinct from, Toxoplasma gondii. N. caninum tachyzoites infect a wide range of mammalian cells both in vivo and in vitro. The mechanisms by which infection is achieved are largely unknown. Recent evidence has suggested that a receptor-ligand system in which one or several host cell receptors bind to one or several parasite ligands is involved. Parasite cell surface-associated molecules such as the recently identified Nc-p43 antigen are prime suspects for being implicated in this physical interaction. In this study it is shown that invasion of Vero cell monolayers by N. caninum tachyzoites in vitro is impaired on incubation of parasites with subagglutinating amounts of affinity-purified antibodies directed against Nc-p43. Postembedding immunogold labeling with anti-Nc-p43 antibodies demonstrated that Nc-p43 is localized not only on the parasite cell surface but also within dense granules and rhoptries. The fate of Nc-p43 during intracellular proliferation of N. caninum tachyzoites and subsequent maturation of the parasitophorous vacuole was also studied.     

Detection of Toxoplasma gondii tachyzoites and bradyzoites in blood, urine, and brains of infected mice.

Different techniques for identifying Toxoplasma gondii were compared. PCR was used to amplify part of the major surface antigen P30 gene of T. gondii. Amplified-DNA detection with the DNA enzyme immunoassay (PCR-DEIA) was more sensitive than ethidium bromide staining after agarose gel electrophoresis and as sensitive as nested PCR. PCR-DEIA, using common enzyme-linked immunosorbent assay (ELISA) methods, avoids agarose gel electrophoresis for the identification of amplified products. T. gondii can also be detected with equal sensitivity in infected fibroblasts, but only after at least 8 days of cell culture. PCR-DEIA is thus recommended because of its sensitivity and convenience for detecting early parasitemia in the surveillance of toxoplasmosis among pregnant women and immunocompromised hosts. The courses of infection in mice infected with two strains of T. gondii were compared. Tachyzoites of the virulent strain T. gondii RH, killing the host in 4 days, were identified in urine specimens and blood samples of mice 24 to 94 h after inoculation but not in brains, but no antibodies were detected. After intraperitoneal inoculation with cysts of the low-level virulence Beverley strain of T. gondii, parasites were identified in blood samples 4 days later and up to 17 days (but not in urine specimens) and in the brain from day 6 through day 525. By ELISA, high antibody titers were found from day 11 to day 525, with parasitemia preceding the appearance of antibodies. The usefulness of PCR-DEIA tests in conjunction with the search for circulating antibodies for the early diagnosis of toxoplasmosis in humans is discussed.     

脂肪干细胞的体外诱导与分化

背景：应用脂肪干细胞来作为组织工程的种子细胞是近年来组织工程研究中的一个活跃领域,大量的研究表明在不同的条件下脂肪干细胞可以向机体的不同组织分化。 目的：对国内外脂肪干细胞体外诱导分化的现状及新进展作一综述。 方法：应用计算机检索CNKI和PubMed数据库中2000-01/2010-10关于脂肪干细胞的文章,在标题和摘要中以“脂肪干细胞,体外分化潜能,种子细胞,转基因”或“Adipose-derived stem cells,cell differentiation in vitro,seeded cell,gene transfaction”为检索词进行检索。选择文章内容与脂肪干细胞体外分化有关者,同一领域文献则选择近期发表或发表在权威杂志文章。初检得到112篇文献,最终入选28篇文献进行综述。 结果与结论：脂肪干细胞是存在于脂肪组织中的具有高度自我更新能力和多向分化潜能的干细胞群体。它具有多向系分化潜能,除可以分化为间充质来源的脂肪、骨、软骨以及骨骼肌、心肌等细胞,也可诱导分化为来源于外胚层的神经细胞以及具有功能性的血管内皮细胞。此外脂肪干细胞还具有造血支持作用以及可被腺病毒等高度转染等优点,可以作为基因转移良好的靶细胞。     

Diagnosis of Sarcocystis cruzi, Neospora caninum, and Toxoplasma gondii infections in cattle

The aim of the study was to diagnose Sarcocystis sp. infections in cattle and to detect coinfections by Toxoplasma gondii and/or Neospora caninum. Blood, diaphragm, esophagus, and myocardium from 90 beef cattle from Argentina were collected. Histopathological, immunohistochemical, polymerase chain reaction assays, and direct microscopical examination were carried out. Sarcocysts from myocardium were measured and counted. Indirect fluorescent antibody test (IFAT) for the three protozoans was performed. Sarcocystis cruzi sarcocysts were found in 100% of myocardium samples. Sarcocysts per gram ranged from 8 to 380 with higher values found in adult cattle (p < 0.001). T. gondii and N. caninum were not detected by immunohistochemistry. T. gondii DNA was found in myocardium of 2/20 seropositive animals, while N. caninum DNA was not found. Antibodies against S. cruzi were detected in all samples, those against N. caninum in 73% and against T. gondii in 91% of the samples (IFAT titer ≥25). It is concluded that serology by IFAT is a suitable method to diagnose these protozoan infections due to its specific IgG detection; therefore, IFAT may be a useful tool to evaluate the impact of each protozoan infection in coinfected animals. Financial support for this study was provided by SeCyT through BID 1728 PICT No. 10858/8.     

骨髓基质干细胞体外定向诱导后表面MHC抗原的表达

背景：同种异体骨髓基质干细胞移植治疗骨骼疾病是研究热点,寻找有效的方法防止移植后免疫排斥反应的发生,是当前迫切需要解决的问题。 目的：探讨骨髓基质干细胞在体外诱导为成骨细胞后表面MHC抗原的表达。 方法：抽取兔骨髓组织,在体外分离、培养获得骨髓基质干细胞,加入含骨形态发生蛋白2重组蛋白的IMDM诱导培养基诱导后,流式细胞仪分析骨髓源性成骨细胞MHC抗原的表达。 结果与结论：骨髓源性成骨细胞表面MHCⅡ类抗原不表达、MHCⅠ类抗原表达。骨髓基质干细胞体外分化为成骨细胞后,未见免疫排斥反应,可用于同种异体或异种骨髓基质干细胞移植治疗骨缺损。 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

体外诱导脂肪干细胞向内皮细胞及成血管的分化

背景：脂肪干细胞作为组织工程中种子细胞的选择,可以诱导为内皮细胞从而有效解决材料血管化困难的难题。 目的：体外观察脂肪干细胞经诱导向内皮细胞转分化的可能性、以及在立体培养基上的血管形成情况。 方法：切取人皮下脂肪,用酶消化法分离和培养脂肪干细胞,将传至第3代或第4代的脂肪干细胞用内皮细胞诱导液、同时在Matrigel三维培养基内诱导培养,观察细胞生长情况及变化。对脂肪干细胞和诱导细胞行免疫组织化学检查CD31的表达。 结果与结论：免疫组织化学检测脂肪干细胞的CD31表达阴性,诱导细胞CD31可见阳性表达。在三维立体培养基内诱导的细胞24 h逐渐迁徙成团,伸出伪足,诱导1周细胞形成网格样交叉,2周形成较长血管,后血管增粗,并出现分叉,CD31阳性表达。因此,脂肪干细胞体外可以被诱导向内皮细胞表型转分化,并形成血管,提示脂肪干细胞可以作为促进组织工程移植物血管化的种子细胞的理想选择。     

In vitro induction and measurement of hemolytic plaque forming cells in man.

Following co-cultivation with sheep red cells or ovalbumin, Hypaque-Ficoll-separated human tonsillar lymphocytes were demonstrated to generate specific hemolytic PFC with maximum numbers at day 5-7. PFC were enumerated on poly-L-lysine coupled red cell monolayers in Microtest-II-plates. Plaque formation appeared to be puromycin-sensitive, complement-dependent and showed clear specificity for the antigen present during the inductive culture. Treatment of PFC with mu-chain specific antisera and complement resulted in complete inactivation of PFC; gamma-chain antisera had no effect. The development of such a simple and sensitive assay system permits the analysis of cellular interactions required for the induction of PFC responses in man.     

体外诱导和检测人类B细胞产生抗HLA抗体的研究

目的 体外诱导和采用酶联免疫斑点法(ELISPOT)检测人类B细胞产生的抗HLA抗体.方法 对7名健康志愿者采用密度梯度离心法分离外周血单个核细胞(PBMC)并体外培养5 d,以B细胞多克隆刺激原美洲商陆丝裂原(PWM)、葡萄球菌A蛋白菌体(SAC)体外刺激PBMC,采用EHSA和ELISPOT方法分别测定培养上清中Ig浓度和抗体分泌B细胞数,探索体外诱导PBMC产生Ig的适宜方法.采用该刺激方法和HLA特异的ELISPOT法,诱导和检测9例拟行肾移植预致敏对象PBMC产生的抗HLA抗体.结果 与PWM单刺激相比,PBMC在PWM和SAC联合刺激后,有更多细胞存活的趋势(P=0.052),且培养上清中IgM的水平明显增加(P=0.03),而IgG的水平无明显变化(P>0.05).6例预致敏对象诱导和检测到抗HLA抗体.其特异性与各自培养上清中检测到的抗HLA抗体一致.结论 PWM和SAC体外刺激PBMC,结合HLA特异的ELISPOT方法,能诱导和榆测到人类B细胞产生的HLA抗体.     

体外诱导和检测人类B细胞产生抗HLA抗体的研究

目的 体外诱导和采用酶联免疫斑点法(ELISPOT)检测人类B细胞产生的抗HLA抗体.方法 对7名健康志愿者采用密度梯度离心法分离外周血单个核细胞(PBMC)并体外培养5 d,以B细胞多克隆刺激原美洲商陆丝裂原(PWM)、葡萄球菌A蛋白菌体(SAC)体外刺激PBMC,采用EHSA和ELISPOT方法分别测定培养上清中Ig浓度和抗体分泌B细胞数,探索体外诱导PBMC产生Ig的适宜方法.采用该刺激方法和HLA特异的ELISPOT法,诱导和检测9例拟行肾移植预致敏对象PBMC产生的抗HLA抗体.结果 与PWM单刺激相比,PBMC在PWM和SAC联合刺激后,有更多细胞存活的趋势(P=0.052),且培养上清中IgM的水平明显增加(P=0.03),而IgG的水平无明显变化(P>0.05).6例预致敏对象诱导和检测到抗HLA抗体.其特异性与各自培养上清中检测到的抗HLA抗体一致.结论 PWM和SAC体外刺激PBMC,结合HLA特异的ELISPOT方法,能诱导和榆测到人类B细胞产生的HLA抗体.     

体外诱导和检测人类B细胞产生抗HLA抗体的研究

目的 体外诱导和采用酶联免疫斑点法(ELISPOT)检测人类B细胞产生的抗HLA抗体.方法 对7名健康志愿者采用密度梯度离心法分离外周血单个核细胞(PBMC)并体外培养5 d,以B细胞多克隆刺激原美洲商陆丝裂原(PWM)、葡萄球菌A蛋白菌体(SAC)体外刺激PBMC,采用EHSA和ELISPOT方法分别测定培养上清中Ig浓度和抗体分泌B细胞数,探索体外诱导PBMC产生Ig的适宜方法.采用该刺激方法和HLA特异的ELISPOT法,诱导和检测9例拟行肾移植预致敏对象PBMC产生的抗HLA抗体.结果 与PWM单刺激相比,PBMC在PWM和SAC联合刺激后,有更多细胞存活的趋势(P=0.052),且培养上清中IgM的水平明显增加(P=0.03),而IgG的水平无明显变化(P>0.05).6例预致敏对象诱导和检测到抗HLA抗体.其特异性与各自培养上清中检测到的抗HLA抗体一致.结论 PWM和SAC体外刺激PBMC,结合HLA特异的ELISPOT方法,能诱导和榆测到人类B细胞产生的HLA抗体.     

体外诱导和检测人类B细胞产生抗HLA抗体的研究

目的 体外诱导和采用酶联免疫斑点法(ELISPOT)检测人类B细胞产生的抗HLA抗体.方法 对7名健康志愿者采用密度梯度离心法分离外周血单个核细胞(PBMC)并体外培养5 d,以B细胞多克隆刺激原美洲商陆丝裂原(PWM)、葡萄球菌A蛋白菌体(SAC)体外刺激PBMC,采用EHSA和ELISPOT方法分别测定培养上清中Ig浓度和抗体分泌B细胞数,探索体外诱导PBMC产生Ig的适宜方法.采用该刺激方法和HLA特异的ELISPOT法,诱导和检测9例拟行肾移植预致敏对象PBMC产生的抗HLA抗体.结果 与PWM单刺激相比,PBMC在PWM和SAC联合刺激后,有更多细胞存活的趋势(P=0.052),且培养上清中IgM的水平明显增加(P=0.03),而IgG的水平无明显变化(P>0.05).6例预致敏对象诱导和检测到抗HLA抗体.其特异性与各自培养上清中检测到的抗HLA抗体一致.结论 PWM和SAC体外刺激PBMC,结合HLA特异的ELISPOT方法,能诱导和榆测到人类B细胞产生的HLA抗体.     

体外诱导和检测人类B细胞产生抗HLA抗体的研究

目的 体外诱导和采用酶联免疫斑点法(ELISPOT)检测人类B细胞产生的抗HLA抗体.方法 对7名健康志愿者采用密度梯度离心法分离外周血单个核细胞(PBMC)并体外培养5 d,以B细胞多克隆刺激原美洲商陆丝裂原(PWM)、葡萄球菌A蛋白菌体(SAC)体外刺激PBMC,采用EHSA和ELISPOT方法分别测定培养上清中Ig浓度和抗体分泌B细胞数,探索体外诱导PBMC产生Ig的适宜方法.采用该刺激方法和HLA特异的ELISPOT法,诱导和检测9例拟行肾移植预致敏对象PBMC产生的抗HLA抗体.结果 与PWM单刺激相比,PBMC在PWM和SAC联合刺激后,有更多细胞存活的趋势(P=0.052),且培养上清中IgM的水平明显增加(P=0.03),而IgG的水平无明显变化(P>0.05).6例预致敏对象诱导和检测到抗HLA抗体.其特异性与各自培养上清中检测到的抗HLA抗体一致.结论 PWM和SAC体外刺激PBMC,结合HLA特异的ELISPOT方法,能诱导和榆测到人类B细胞产生的HLA抗体.